| Design Description | The growth of PBCV-1 host C. variabilis NC64A on MBBM medium and the production and purification of PBCV-1 have been described (Van Etten et al., 1983). 3 x 109 exponentially growing cells were pelleted by centrifugation after exposure to PBCV-1 (MOI = 5). Cells were immediately flash frozen in liquid nitrogen and stored at -80 *C until further processing. The durations of infection lasted for 7, 14, 20, 40, and 60 min. Cells for the 0 min. p.i. time point (i.e., healthy) were not exposed to PBCV-1 until after flash freezing. The RNA-seq library was constructed from 10 mg of total RNA extracted for each time point using the mRNA-seq Sample Preparation Kit (RS-100-0801) according to the manufacturer's instructions (Illumina). RNA was subjected to poly(A) selection using Sera-Mag Magnetic Oligo-dT Beads followed by fragmentation and then used for cDNA synthesis with random hexamers. The cDNA product then underwent end repair, A-tailing, adapter ligation, and PCR amplification. Each library was sequenced using an Illumina GAIIX sequencer on one lane of the flow cell, generating 15.8 to 19.7 million 51-nt single-end reads for each time point. |
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